The long-term goal of the proposed project is to understand the functional significance of ribonucleoside modifications. In spite of the large number of studies which have identified over 90 modifications and their locations in tRNAs and larger RNA molecules, little is known regarding the individual contributions of these modifications to RNA structure and function. We hypothesize that modified nucleosides exert their effects on RNA function by altering the stability or structure at specific sites within the RNA. Thus, this project is to determine the specific contributions of modified nucleosides to RNA structure and stability. The inability to incorporate modified nucleosides in a site- specific manner into large RNAs has complicated both structural and functional analyses of these molecules. The specific aims of this proposal are 1) to develop methodologies for site-specific incorporation of modified nucleosides into RNA and 2) to determine the effects of individual modified bases on structural stability of both ribonucleotide fragments (model RNAs) and of full-length RNA molecules with specific biological functions. The site-specific incorporation has to be both versatile and adaptable to various modifications. Therefore, two complementary approaches are proposed: the first involves phosphoramidite chemistry in which large quantities of synthetic RNAs can be obtained in a routine manner; the second involves a more general, enzymatic incorporation that is applicable to all of the modified bases and to much larger, biologically interesting RNA molecules. These studies will allow us to understand the unique effects of the individual modified nucleotides on RNA structure and function. The ability to modify RNA at specific sites is important for determining structure-function relationships between RNA and proteins or small molecules, effects on folding and dynamics, and metal-binding effects of modified bases. RNA is the center of much attention because of its ability to catalyze specific reactions. Modified RNAs with enhanced catalytic activities may also have potential therapeutic applications.